D. Boulanger

Characterization of Monoclonal Antibodies to Bovine Viral Diarrhoea Virus: Evidence of a Neutralizing Activity against Gp48 in the Presence of Goat Anti-Mouse Immunoglobulin Serum

Twenty-one monoclonal antibodies (MAbs) directed against the NY-1 and the Osloss-c strains of bovine viral diarrhoea virus were produced and characterized by indirect immunofluorescence assay, radioimmunoprecipitation and neutralization tests. Fourteen MAbs directed against the NY-1 strain recognized the gp48 and showed a weak neutralizing activity in the presence of goat anti-mouse immunoglobulin serum.

Serological survey for orthopoxvirus infection of wild mammals in areas where a recombinant rabies virus is used to vaccinate foxes.

Several fox vaccination campaigns against rabies have been undertaken in Belgium by using a vaccinia-rabies recombinant virus distributed in baits in the field. However, foxes and other wild animals that may ingest the baits could be infected at the same time by another orthopoxvirus, such as cowpox virus, which circulates in wildlife. Recombination between the two viruses could therefore occur. A serological survey for antibodies to orthopoxvirus, and particularly to cowpox virus, was undertaken in foxes and in several other wild species. Antibodies were detected only in two rodent species, in 16 of 25 bank voles (64 per cent) and in two of 29 woodmice (7 per cent). The risk of virus recombination in wildlife can therefore be considered to be extremely low.

Proteins specified by bovine herpesvirus type 4: structural proteins of the virion and identification of two major glycoproteins by using monoclonal antibodies

Bovine herpesvirus type 4 proteins were identified by PAGE of [35S]methionine- or [3H]glucosamine-labelled purified virions. Thirty-one monoclonal antibodies (MAbs) raised against the V. Test strain were used to identify 29 proteins, ten of which were glycosylated. All of these glycoproteins belonged to the viral envelope and a 140K non-glycosylated protein appeared to be the major nucleocapsid protein. The MAbs were classified into two groups. The first group precipitated three glycoproteins of Mr 150K, 120K and 51K. The 120K and 51K glycoproteins were linked by disulphide bonds and the 150K glycoprotein was linked to the others by non-covalent bonds. The second group precipitated a different 120K glycoprotein.

A monoclonal ELISA for bovine viral diarrhoea pestivirus antigen detection in persistently infected cattle.

Detection of cattle persistently infected with bovine viral diarrhoea virus (BVDV) is crucial to controlling mucosal disease. A sandwich enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies raised against the 48-kDa glycoprotein and the 120/80-kDa protein was developed for detecting antigens in leucocytes of 3 persistently BVDV-infected calves. The test is simple, sensitive and rapid. Moreover the same ELISA was able to recognise Belgian field isolates of BVDV. These results show that the test can be applied in the field.

Production and characterization of monoclonal antibodies to bovid herpesvirus-4

Thirty-five hybridoma cell lines secreting monoclonal antibodies (Mabs) against bovid herpesvirus-4 (BHV-4) strain V. Test were produced. These hybrid cells resulted from the fusion of SP2/0 myeloma cells with splenocytes of BALB/c mice previously immunized with purified BHV-4. A modified indirect fluorescent antibody test (IFAT) was applied as a screening procedure and was compared with an indirect enzyme-linked immunosorbent assay. The selected Mabs were tested by the same IFAT against a panel of BHV-4 field isolates and against bovine herpesvirus-1, bovine herpesvirus-2 and alcelaphine herpesvirus-1 (AHV-1). Comparison of BHV-4 field isolates with Mabs confirmed their close antigenic relationships, but slight antigenic differences were observed between different isolates. One of the Mabs also reacted against AHV-1, indicating an antigenic relationship between BHV-4 and AHV-1. None of the Mabs reacting with BHV-4 possessed neutralizing activity against the strain used for immunization.

Epidemiological evaluation of a monoclonal ELISA detecting bovine viral diarrhoea pestivirus antigens in field blood samples of persistently infected cattle

An enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies for capture and detection, was developed for detecting bovine viral diarrhoea virus (BVDV) antigens in blood samples. The test was evaluated using 761 field samples of known status (viraemic or not). When an appropriate cut-off value was chosen, the sensitivity, specificity, and predictive values of the assay were 100%, higher than the values obtained by classical virus isolation. Correlation with the latter technique exceeded 90%. The ELISA is a good candidate for replacing virus isolation as a reference method for BVDV antigen detection in persistently infected carriers. A method based on the mean of the standard deviation ratio can be used to choose the cut-off value in order to optimise reproducibility.

Comparison of the susceptibility of the red fox (Vulpes vulpes) to a vaccinia-rabies recombinant virus and to cowpox virus

Sylvatic rabies can be efficiently controlled by vaccination of foxes with a vaccinia-rabies recombinant virus. However, the risk of recombination between the engineered vaccine virus and other orthopoxviruses endemic in wildlife, such as cowpox virus, still needs to be investigated. In this study, foxes inoculated orally and intradermally with cowpox virus were found to be not very susceptible to cowpox virus, which was isolated from only the oropharynx and tonsils, at low titre and for only five days after inoculation. Thus the risk of recombination between these viruses in foxes is very low.

Neutralization of Bovine Herpesvirus Type 4 by Pairs of Monoclonal Antibodies Raised against Two Glycoproteins and Identification of Antigenic Determinants Involved in Neutralization

In infected cattle, bovine herpesvirus type 4 (BHV-4) induces an immune response with low neutralizing antibody levels or in the absence of such antibodies. For the study of this phenomenon, monoclonal antibodies (MAbs) raised against two BHV-4 glycoproteins identified previously (150K/120K/51K and 120K/16.5K) were used in neutralization tests. None of the MAbs except for MAb 16 could neutralize alone; pairs of MAbs against the 150K/120K/51K and 120K/16.5K glycoproteins were able to neutralize BHV-4 infectivity. MAbs involved in neutralization were used in competitive binding assays to identify epitopes relevant for BHV-4 neutralization. These MAbs showed a low avidity and a weak neutralizing activity, and they partially decreased BHV-4 attachment to cells. These results suggest that the BHV-4 glycoprotein domains involved in viral infectivity are poorly exposed to the immune system.

Field trials of a recombinant rabies vaccine.

To improve both safety and stability of the vaccines used in the field to vaccinate foxes against rabies by the oral route, a recombinant vaccinia virus, expressing the glycoprotein of rabies virus (VVTGgRAB) has been developed. VVTGgRAB innocuity was verified in target species and in domestic animals as well as in numerous wild animal species that could compete with the red fox in consuming vaccine baits in Europe. Oral immunization of foxes, by distributing VVTGgRAB vaccine-baits, was undertaken for the whole infected area in Belgium (10,000 km2). Five campaigns of fox vaccination, were carried out from autumn 1989 until 1991. Each time, 150,000 vaccine-baits were dropped by air at a mean density of 15 per km2. These campaigns induced a drastic decrease in the incidence of rabies and the elimination of the disease from 80% of the initially infected area. Regarding the geographical evolution of rabies in Belgium and in adjacent regions in neighbouring countries, new spatial strategies for bait dispersal were planned for 1992, 1993 and 1994: successive confined campaigns were carried out along political borders only. These campaigns induced a new decrease of incidence; no rabid fox could be detected in 1993 in spite of an improved epidemiological surveillance. In 1994, rabies was again confirmed in 13 foxes collected in an area close to the French border. These cases demonstrated the persistence of a border rabies focus and justify further restricted vaccination campaigns.

Rabies Recombinant Vaccines: Development and Field Application

Rabies is one of the oldest recognized diseases. It most probably originated on the African continent and diffusion in North Africa was followed by the spread throughout Europe and Asia. The disease subsequently appeared on the North American continent possibly by transmission through circumpolar animals such as foxes and wolves, though the importation of rabid dogs from Europe is also probable. Today rabies is a disease of major importance, present on all five continents (Theodorides, 1986; Kieny et al., 1987; Kurstak, 1993).